Cancers circumvent immune surveillance via production of transforming growth factor-? (TGF-?), a multifunctional cytokine that facilitates tumor progression, invasion, and metastasis via promotion of epithelial- mesenchymal transition (EMT) and suppression of immune responses. TGF-? directly attenuates the activation and cytotoxic function of immune effector cells (NK cells and CD8+ T cells), and promotes the development and function of regulatory T cells (Tregs), a sub-population of immunosuppressive CD4+ T cells that express forkhead box P3 (FoxP3+). TGF-? induces expression of FoxP3, which upregulates cytotoxic T lymphocyte antigen-4 (CTLA-4), a molecule that restrains the activation of T cells. Our studies demonstrate that TGF-? and CTLA-4 function as cooperative, yet redundant, brakes on the immune system, and indicate that the abrogation of these immune checkpoints is required to counteract Tregs and unleash innate and adaptive immune responses against tumor cells. To advance this strategy, we have developed two novel bifunctional immunostimulatory antibodies to counteract TGF-? in the microenvironment of the targeted tumor cell or Treg: 1. Anti-EGFR-TGF?RII: A bifunctional antibody that sequesters TGF-? via an extracellular domain sequence of TGF Receptor II fused to the C-terminus of the heavy chain of anti-EGFR antibody (Cetuximab). 2. Anti-CTLA4-TG?RII: A bifunctional antibody that sequesters TGF-? via an extracellular domain sequence of TGF-? Receptor II fused to the C-terminus of the heavy chain of anti-CTLA-4 antibody (Ipilimumab). Approximately 15-25% of patients with breast cancer have triple negative breast cancer (TNBC), an aggressive treatment-refractory subtype that lacks hormone receptors and HER2/neu. Although 80% of TNBC overexpress epidermal growth factor receptor (EGFR), they do not respond to cetuximab. Our studies demonstrate that TGF-? is a key determinant of the resistance of cancers to cetuximab. Since TNBC exhibit EMT and Treg infiltration, the tell-tale signs of TGF-? activity, they may be ideal targets for targeted bifunctional immunostimulatory antibodies that counteract TGF-?-mediated immune tolerance. The Specific Aims are: I. Evaluate the antitumor efficacy of anti-EGFR-TGF?RII in TNBC tumor-bearing mice: (A) Examine whether anti-EGFR-TGF?RII has superior antitumor efficacy compared to cetuximab, and determine the role of ADCC in its mechanism of action; (B) Examine whether anti-EGFR-TGF?RII can reverse immune tolerance and enhance T cell-mediated tumor regression in NOG mice reconstituted with human CD34+ cells. II. Evaluate the antitumor efficacy of anti-CTLA-4-TGF?RII in TNBC-bearing NOG mice reconstituted with human CD34+ cells: (A) Examine whether anti-CTLA-4-TGF?RII counteracts Tregs and activates antitumor immunity; (B) Compare the antitumor efficacy of anti-CTLA-4-TGF?RII with that of Ipilimumab or anti-TGF-?. IMPACT: The project seeks to advance the treatment of TNBC and other common cancers by counteracting tumor-induced immune tolerance via novel targeted bifunctional immunostimulatory antibodies.